Eukaryotic cells from yeasts to human utilize multiple MAP kinase (MAPK) cascades to transmit diverse extracellular stimuli to the nucleus. Among these are MAPKs dedicated for stress signaling, which are also called SAPKs (Stress-Activated Protein Kinases). Accumulating evidence strongly suggests that the functions of human SAPKs are of clinical importance, especially in control of cell death and proliferation, the inflammation response and cell differentiation. In addition, SAPKs appear to be key determinants of the response of tumor cells to cytotoxic treatments and chemotherapeutic drugs. The long-term objective of the proposed research is to attain a comprehensive understanding of SAPK regulation in response to cytotoxic stress stimuli, an understanding which is crucial to the control of SAPKs in the clinical context. These studies will be carried out in the genetically tractable model system provided by the fission yeast Schizosaccharomyces pombe. This organism has been used successfully for discovering and analyzing basic biological mechanisms that are conserved among eukaryotes. Our previous studies have demonstrated that both the structure and function of the SAPK pathways are highly conserved between fission yeast and human. Particular aspects of this conservation which are important for this proposal include the facts that in both organisms the SAPK pathways respond to multiple and diverse stresses and control the activity of an ATF transcription factor. Therefore, general principles of SAPK regulation and stress signaling in eukaryotes should emerge from the proposed research. The goal of this project is to elucidate the mechanisms which transmit diverse environmental stress signals to the S. pombe SAPK, Spc1. The specific aims focus on key upstream components of the Spc1 pathway, including the Wis1 MEK, the Wis4/Win1 MEKKs, and Mcs4, a homolog of the "response regulator" member of bacterial two-component systems. Genetic and biochemical experiments will determine how MEKKs and the two-component signaling system regulate the Wis1 MEK in response to diverse stresses, including osmostress, oxidative stress, heat shock and UV irradiation. Additional regulatory mechanisms independent of Wis4/Win1 and Mcs4 will also be sought by genetic screens. Lastly, recent experiments strongly suggest that the Pyp1 and Pyp2 tyrosine phosphatases, which negatively regulate the Spcl SAPK, are directly involved in heat stress signaling to Spc1. This mechanism will be investigated in detail.